Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Abstract

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Figure 1

A phylogenetic tree of putative NTPase genes of type II/IV secretion systems constructed with maximum parsimony algorithms. Gene subfamilies are designated by colored boxes, and subgroupings, by vertical lines. Archaeal and Bacterial divisions in the tadA gene subfamily are shown in two shades of green. The numbers on branches are Bremer indices of the nodes at the end of the designated branch (see Methods). Bremer indices for some organisms did not fit onto the tree and are supplied in the box in the upper left corner. Accession numbers for all genes are listed on the web site http://cpmcnet.columbia.edu/dept/figurski.

Figure 2

Scheme for rooting the tree. The network shows the two divisions in the superfamily tree between Bacteria and Archaea. Archaeal groups are shown in shaded boxes. Shaded circles show roots that would conserve likely splits between the Archaea and Bacteria. Single solid circles represent roots that maintain a phylogenetic difference between type II and IV secretion NTPase genes. The double solid circle creates a root that unites all proteins with all four conserved domains (Walker A and B, and the His and Asp boxes).

Figure 3

Diagnostic key for classification of putative NTPase genes of type II/IV secretion. Starting with the diagnostic sequences for type II/IV putative NTPases, one can place the newly discovered gene into a family, subfamily, and, in some cases, subgroup. Numbers represent standardized positions on the MPASs. Any one of the amino acids listed after the position number is diagnostic for that position. The boxes represent major monophyletic groups in the tree. To advance to the next box, a sequence must contain a diagnostic amino acid at each position listed in that box. Our web page (http://cpmcnet.columbia.edu/dept/figurski) performs the diagnosis and classifies new genes.